Immunology 1977 32 247

Effects of cholera exotoxin on Fc receptor activity of lymphoid cells and mononuclear phagocytes

S. H. ZUCKER MAN & S. D. DOUGLAS Departments of Medicine and Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota, U.S.A.

Received 11 March 1976; accepted.for publication 29 July 1976

toxicity (Perlmann, Perlmann & Wigzell, 1972; Michaelsen, Wisloff & Natvig, 1975) in the humoral immune response (Unanue, 1975) in opsonization and phagocytosis (Mantovani, Rabinovitch & Nussenzweig, 1972; Huber, Douglas & Fudenberg, 1969). Fc receptors for IgG occur on several mammalian cell types (Boyden & Sorkin, 1960; Huber & Fudenberg, 1968; and LoBuglio, Cotran & Jandl, 1967) which include blood monocytes, splenic cells and macrophages, neutrophils (Gale & Zighelboim, 1974), B cells (Basten, Miller, Sprent, & Pye, 1972; Dickler & Kunkel, 1972), activated T cells (Anderson & Grey, 1974) and other non-lymphoid cell types [e.g., cytomegalovirus infected fibroblasts (Keller, Peitchel, Goldman & Goldman, 1976)]. Although the IgG subclass and domain specificity has been studied in various systems (Huber, Douglas, Nusbacher, Kochwa & Rosenfield, 1971; Froland, Michaelsen, Wisloff & Natvig, 1974) and preliminary fractionation of the receptor has been reported from murine splenic cells (Rask, Klareskog, Ostberg & Peterson, 1975), the mechanisms of intracellular regulation of Fc receptors and possible differences in Fc receptors between lymphoid and phagocytic cell types have not been characterized. Rhodes has reported that the Fc receptors on guinea-pig macrophages are heterogenous in binding affinities for antibody coated erythrocytes (Rhodes, 1975a). Arend & Mannik (1973) using soluble antigen-antibody complexes have demonstrated that 'activated' rabbit alveolar macrophages have more receptor sites with increased binding affinity for

Summary. The effect of cholera exotoxin and aminophylline on Fc receptors in a murine lymphoidcell line and in rabbit pulmonary alveolar macrophages has been investigated. Although both agents elevated intracellular cyclic AMP levels in macrophages and lymphoid cells, the effects on Fc receptor expression were distinct. Cholera toxin at 10 pg/ml reversibly inhibited Fc-receptor activity in the murine lymphoid cell line. In contrast, cholera toxin at 10 ,ug/ml or 0 01 ug/ml was ineffective in altering pulmonary alveolar macrophage receptor expression. Fc receptor activity on the macrophage was reduced by 20-30 per cent following incubation with aminophylline, (10- 3 M) from 0-6 h. There was no direct correlation between Fc-receptor activity and cyclic AMP levels in the cells studied. The differential susceptibility of these lymphoid and phagocytic cell populations to cholera toxin and also toward aminophylline suggests that there may be fundamental differences in topography on the membrane surface, or in the intracellular regulation of Fc receptors between lymphoid and phagocytic cells. INTRODUCTION Fc receptors have been considered to have an important role in antibody-dependent cell-mediated cytoCorrespondence: Dr Steven D. Douglas, Professor of Medicine and Microbiology, University of Minnesota Medical School, Mayo Memorial Building Box 1, Minneapolis, Minnesota 55455, U.S.A. C

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S. H. Zuckerman & S. D. Douglas

these complexes, than have non-activated cells. We have recently demonstrated that at high doses, cholera toxin, a potent activator of adenylate cyclase, inhibits Fc receptor activity in a murine lymphoid cell line (Zuckerman & Douglas, 1975). Following exposure to cholera exotoxin, freeze fracture of these cells revealed an increase in intramembranous particles (IMP) primarily on the extracellular face (B face) of the plasma membrane (Douglas, Ooka, & Zuckerman, 1976); electron microscopy of thin sections showed large amplitude mitochondrial swelling (Douglas, Zuckerman & Ooka, 1976). The role of cyclic nucleotides in Fc receptor expression is unknown. Agents which increase intracellular cyclic AMP have been demonstrated to inhibit a number of immunologic events which include mitogen stimulation (Whitfield, Rixon, MacManus & Balk, 1973), lysosome degranulation (Zurier, Weissmann, Hoffstein, Kammerman & Tai, 1974), histamine release (May, Levine & Weissmann, 1970), T-lymphocyte rosette formation (Chisari & Edgington, 1974) and antibody-dependent cellmediated cytotoxicity (Gale & Zighelboim, 1974). In an attempt to further characterize Fc receptors in lymphoid and phagocytic cells, we have performed studies of the effects of cholera toxin and aminophylline on murine lymphoid cells and rabbit pulmonary alveolar macrophages (PAMs). These studies indicate that macrophage Fc receptors in contrast to murine lymphoid cells, are insensitive to toxin-mediated inhibition. Fc receptor activity can, however, be partially inhibited following, incubation of PAMs with aminophylline. MATERIALS AND METHODS Cell source A murine lymphoblastoid cell line, derived from a spontaneous myeloma in the BALB/c mouse, PU-5 was obtained from Dr Joel Buxbaum (New York University Medical School) and has been maintained in our laboratory as a suspension culture for 2 years. This cell line (Fc +) has a plasmamembrane receptor for the Fc portion of IgG and is maintained at 370 in a 5 per cent CO2 atmosphere at a concentration of 1-2 x 106 per ml in Dulbecco's modified essential medium with 20 per cent horse serum supplemented with antibiotics, glutamine, and non-essential amino acids. Pulmonary alveolar macrophages were obtained from 3-4 kg male

albino rabbits, killed by air embolism. Macrophages were removed by tracheal lavage of the lungs with heparinized saline (Myrvik, SotoLeake, & Farris, 1961; Daughaday & Douglas, 1976). Yields varied between 60-90 x 106 cells per rabbit, 90 per cent of which were macrophages. Cells were washed in TC-199 and resuspended at concentrations of 1-2 x 106/ml in RPMI-1640 with 15 per cent foetal calf serum. Receptor Assay Fc-receptor activity was assayed using sheep erythrocyte suspension coated with a 1:400 dilution of rabbit 7S Forssman antibody (EA). For the Fc + line, an equal volume of 0-5 per cent EA was added to the cell suspension, incubated at 370 for 30 min and the number of rosettes (cells binding three or more sensitized erythrocytes) was determined for 200 cells. In studies with PAMs, 1-2 x 106 macrophages were incubated at 370 for 1 h in Leighton tubes to permit adherence to glass coverslips. PAMs were then exposed to cholera toxin or aminophylline and further incubated for 0-6 h. Following incubation, Leighton tubes were washed twice with TC-199, resuspended in TC-199 and 01 ml of 5 per cent EA was added (approximately 150-250 erythrocytes per macrophage) and incubated for 1 h. Coverslips were removed, washed in TC-199, fixed in methanol and stained with Wright-Giemsa with Clark and Lubs phosphate buffer and mounted. Two hundred cells per coverslip were scored for rosette formation, phagocytosis, or combined interaction, (percent

positive activity). Experimental design Cholera exotoxin (Lot 0172) prepared by R. A. Finkelstein (Finkelstein & LoSpalluto, 1970) under contract for the National Institute of Allergy and Infectious Diseases was obtained from C. Miller. Cholera exotoxin at final concentrations of 0 01 ,ug/ ml and 10 4g/ml was incubated with both cell types and at various times cell samples were removed and assayed for receptor activity. Aminophylline (Sigma Chemical Co.) was used at a final concentration of 10-3 M. When both toxin and aminophylline were used together with the Fc + line, they were added at time zero. Aminophylline was added to toxintreated macrophages 1, 2 and 4 h after exposure to toxin for the I i, 3- and 6-h time points, respectively. Cyclic AMP was determined on cell suspensions of 10-20 x 106 cells. Cell suspensions were centrifuged

249

Fc receptor activity of lymphoid cells

tion of intracellular cAMP and yet did not affect Fc receptor expression. The effect of exotoxin on receptor activity was evident immediately following the

at 800 g at 40 and cell pellets extracted with 0 05 sodium acetate, pH 4 0, at 800 (DeRubertis,

M

Zenser, Adler & Hudson, 1974). Quantification was performed on triplicate samples at each time point using the radioimmunoassay procedure of Steiner Parker & Kipnis (1972) (Schwarz-Mann).

addition of toxin (Fig. 1). Inhibition

was

still

apparent at 12 h and receptor activity, despite the continued presence of toxin, returned to control values at 24 h. Aminophylline alone had no significant effect on receptor activity (data not shown) and

in the presence of exotoxin, aminophylline slightly potentiated toxin induced receptor inhibition at 24 h. In contrast to the observed inhibition of receptor activity in the Fc + line, we were unable to demonstrate an inhibitory effect of the toxin (10 pg/ml or 0 01 ,g/ml) on the macrophage Fc receptor (Table 1).

RESULTS Cholera exotoxin (10 ,ug/ml), as we have previously demonstrated (Zuckerman & Douglas, 1975), has a potent inhibitory effect on receptor activity in the PU-5 Fc+ lymphoid cell line. At the lower concentration (0 01 jg/ml), cholera toxin caused eleva-

80 70 601-

50 40 30 20 10

.]II 0

3

Th

6

12

24

30

Time (h) Figure 1. Effect of aminophylline and cholera exotoxin on murine lymphoblastoid Fc receptor. The mean number of rosettes/200 cells for four experiments is plotted on the ordinate. Standard deviation+ four to eight rosettes. Hatched columns, control; open columns, cholera toxin 0 jug/ml; stippled columns, cholera toxin 10 ,ug/ml+ aminophylline.

Table 1. Effect of cholera toxin on macrophage Fc receptor and cAMP levels

Time (h)

0

6

3

Per cent positive activity

cAMP levels*

Per cent positive activity

cAMP levels

Per cent positive activity

cAMP levels

83-4+ 10-0 84-8+ 99 83-1+11-1

1-85+ 008 n.d. n.d.

78-9+119 80-0+ 10-7 80-8+ 8-1

1-54+ 0-11 2-79+0-12 2-81+0-09

74-3+ 15 74-7+ 13-4 74-4+10-3

n.d. n.d. n.d.

Toxin (concentration) 001

Ag/ml

lOpcg/ml

Mean values obtained for percent positive activity and cAMP levels were based on six and three experiments, respectively. cAMP values at 3 h for 10 pg and 0 01 ug as compared to control were significant P< 0 001. n.d. = Not done. *p mole/ 106 cells.

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S. H. Zuckerman & S. D. Douglas

Both concentrations of toxin, following incubation for 3 h, resulted in increased intracellular cAMP levels. Aminophylline alone inhibited receptor activity for glass adherent macrophages at concentrations greater than 10-5 M and this inhibition became more significant with increasing concentrations (Fig. 2). In further studies we used aminophylline at 10-3 M. Although greater inhibition of receptor activity is apparent at higher concentrations, there appeared to be an inverse correlation between the concentration of aminophylline and the number of glass-adherent macrophages. The viability, as assessed by trypan blue dye exclusion in the presence of 10-3 M aminophylline, or in control Leighton tubes, was greater than 95 per cent for non-adherent cells. Aminophylline inhibited receptor activity by 20-30 per cent when incubated with macrophages from 0-6 (Fig. 3). This inhibitory effect was reversible following the removal of aminophylline and receptor activity approached control values within the 1-h incubation with EA, required for receptor assay (data not shown). Incubation with cholera toxin, at both high and low doses, in combination with aminophylline (10-3 M) resulted in receptor inhibition which was comparable to that with aminophylline alone (Fig. 3). The difference observed for the inhibition by toxin plus aminophylline, compared to aminophylline alone, was of statistical

significance only at 90 min (P< 0 025). The inhibition in receptor activity observed at both 3 and 6 h was associated with elevation of intracellular cAMP (Table 2). However, at both 3 and 6 h the intracellular cAMP levels were greater in macrophages incubated with toxin (0 01 ug/ml) alone than for cells incubated with aminophylline. Also, despite the comparable degree of receptor inhibition for macrophages treated with toxin and aminophylline, compared to aminophylline alone, the former had significantly greater intracellular cAMP values (Table 2) DISCUSSION Our findings indicate that Fc receptor expression in different cell types may be influenced by distinct regulatory processes. Cholera exotoxin at a concentration of 10,ug/ml inhibited Fc receptor activity in the lymphoblastoid cell line in a reversible manner (Fig. 1). Using aminophylline in combination with cholera toxin, we were unable to demonstrate further significant receptor inhibition. The relationship of toxin-mediated cAMP elevation to receptor inhibition is unknown, as cholera toxin at 0 01 ,g/ml elevated cAMP to values comparable with 10 pg/ml but had no effect on receptor expression (Zuckerman & Douglas, 1975).

001go

k

80 F 70 H

60 [-

0

50 F-

40F30 F 20 H 10 1,,,11

io-6

10-5

I I

I

I,,

I

io-4

III

I I

1I ,IIII

i0-3

o0-2

Aminophylline

Figure 2. Effect of aminophylline on pulmonary alveolar macrophage Fc receptor. The percent positive cells is plotted function of the molarity of aminophylline. The control Fc activity is the upper point on the y-axis (98 per cent).

as a

Fc receptor activity of lymphoid cells

251

Time(h)

Figure 3. Effect of aminophylline and cholera exotoxin on rabbit pulmonary alveolar macrophage Fc receptor. Y axis represents percent positive activity. Brackets are one standard deviation. P< 0-025 compared to control with aminophylline from 0-1 1/2 h and OOOP. 0.01 for all other values, four experiments. Hatched columns, control; stippled columns, aminophylline, open columns, toxin 10 ,ug/ml+aminophylline; cross-hatched columns, toxin 0-01 ug/ml+aminophylline. Table 2. Effects of cholera exotoxin and aminophylline on rabbit PAM cAMP levels*

Time (h)

Control

Aminophylline

Toxin 0 01 pg/ml

Toxin 0-01 ug/ml + aminophylline

3 P< 6 P